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sbh00 | ACSJCA | JCA10.0.1465/W Unicode | research.3f (R3.4.i1:3887 | 2.0 alpha 39) 2012/09/13 09:54:00 | PROD-JCA1 | rq_718674 |
10/15/2012 07:12:42 | 9
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pubs.acs.org/acschemicalbiology
A Short DNA Aptamer That Recognizes TNFα and Blocks Its Activity
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Erik W. Orava,†,§ Nick Jarvik,∥ Yuen Lai Shek,† Sachdev S. Sidhu,∥ and Jean Gariépy*,†,‡,§
†
Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario M5S 3M2, Canada
Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 2M9, Canada
§
Physical Sciences, Sunnybrook Research Institute, 2075 Bayview Avenue, Toronto, Ontario M4N3M5, Canada
∥
Terrence Donnelly Center for Cellular and Biomolecular Research, and Banting and Best Department of Medical Research,
Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 3E1, Canada
‡
S Supporting Information
*
ABSTRACT: Tumor necrosis factor-alpha (TNFα) is a pivotal component
of the cytokine network linked to inflammatory diseases. Protein-based,
TNFα inhibitors have proven to be clinically valuable. Here, we report the
identification of short, single-stranded DNA aptamers that bind specifically
to human TNFα. One such 25-base long aptamer, termed VR11, was shown
to inhibit TNFα signaling as measured using NF-κB luciferase reporter
assays. This aptamer bound specifically to TNFα with a dissociation constant
of 7.0 ± 2.1 nM as measured by surface plasmon resonance (SPR) and
showed no binding to TNFβ. Aptamer VR11 was also able to prevent
TNFα-induced apoptosis as well as reduce nitric oxide (NO) production in cultured cells for up to 24 h. As well, VR11, which
contains a GC rich region, did not raise an immune response when injected intraperitoneally into C57BL/6 mice when compared
to a CpG oligodeoxynucleotide (ODN) control, a known TLR9 ligand. These studies suggest that VR11 may represent a simpler,
synthetic scaffold than antibodies or protein domains upon which to derive nonimmunogenic oligonucleotide-based inhibitors of
TNFα.
U
mechanisms to local injury but can also cause acute and chronic
tissue damage.11,12
Despite advances in antibody and protein engineering, the
major drawbacks of protein-based TNFα inhibitors are their
immunogenicity arising from their chronic use and their
production costs resulting in expensive therapies for patients.
Simpler, synthetic, nonimmunogenic classes of TNFα inhibitors could be derived from short, single-stranded nucleic acid
oligomers (ssDNA or RNA) known as aptamers. These
molecules adopt a specific tertiary structure allowing them to
bind to molecular targets with high specificity and affinities
comparable to those of monoclonal antibodies.13 In some cases,
aptamers will display functional properties beyond just binding
to their target. For instance, an aptamer to the inflammation
factor human neutrophil elastase (hNE) was shown to
significantly reduce lung inflammation in rats and displayed
greater specificity for their target than an antielastase IgG
control.14 Examples of other aptamers exhibiting functional
attributes include a DNA aptamer to anti-HIV reverse
transcriptase and RNA aptamers to the basic fibroblast growth
factor and vascular endothelial growth factor.15−17 Finally, a
single-stranded DNA aptamer selected to bind to thrombin has
nregulated immune responses are intimately associated
with degenerative diseases such as atherosclerosis,
arthritis, encephalitis, and tumors.1−3 The primary proinflammatory cytokine, tumor necrosis factor alpha (TNFα),
plays a critical regulatory role in enhancing these responses.4 In
the past decade, disease-modifying antirheumatic drugs
(DMARDs) that target underlying immune responses
processes have improved disease outcomes in patients with
chronic immune response disorders.5 Anti-TNFα protein-based
therapies (Enbrel, Humira, InfliximAb) have emerged as a
dominant category of DMARDs.6 However, ∼40% of patients
still display moderate to high levels of disease even after
treatment with protein therapeutics, suggesting a substantial
need for improved therapies.7
TNFα is a pro-inflammatory cytokine that is produced by an
array of cell types such as macrophages, monocytes,
lymphocytes, keratinocytes, and fibroblasts in response to
inflammation, infection, injury, and other environmental
challenges.8 TNFα is a type 2 transmembrane protein with
an intracellular amino terminus and is synthesized as a 26-kD
membrane-bound protein (pro-TNFα) that is cleaved to
release a soluble 17-kD TNFα molecule. TNFα has the ability
to signal as a membrane-bound protein as well as a soluble
cytokine. As a soluble cytokine, TNFα is active only as a
noncovalently associated homotrimer.9,10 Sufficient levels of
TNFα as well as other mediators are critical for sustaining
normal immune responses. TNFα can initiate host defense
© XXXX American Chemical Society
Received: March 15, 2012
Accepted: October 9, 2012
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been shown to inhibit thrombin-catalyzed fibrin-clot formation
in vitro using either purified fibrinogen or human plasma.18
In the present study, we constructed a 25-nucleotide variable
region DNA library to perform systematic evolution of ligands
by exponential enrichment (SELEX)19 selections using
recombinant TNFα as our target. Several ssDNA aptamers
shown to specifically bind to TNFα were further evaluated for
their ability to inhibit TNFα signaling in several independent in
vitro assays. These analyses led to the identification of VR11, a
DNA aptamer capable of inhibiting TNFα functions in vitro.
■
RESULTS AND DISCUSSION
Identification of DNA Aptamers Directed at TNFα.
Traditional treatments for chronic inflammation include
NSAIDs, glucocorticoids, cytostatic drugs and DMARDs. Due
to the increased understanding of the molecular pathology of
several inflammatory disorders, immunosuppressants, such as
those directed at TNFα, have moved to the forefront of antiinflammatory drugs. The current anti-TNFα therapy market is
dominated by protein therapeutics, namely, etanercept
(Enbrel), adalimumAb (Humira), and infliximAb (Remicade).
These TNFα inhibitors have been shown to be therapeutically
non-equivalent in terms of their structure, binding to TNFα,
and their therapeutic effects.20 More importantly, these proteinbased therapeutics are large macromolecules that are costly to
produce and immunogenic when used in a chronic therapy
setting, with close to 40% of patients still showing disease
symptoms even after treatment.
Using TNFα as a target, we perform SELEX searches with a
view to identify short, synthetic DNA aptamers able to
recognize this key cytokine and potentially mimic the inhibitory
action of an anti-TNFα antibody (Figure 1). After 12 rounds of
selection, 60 clones were sequenced and found to be enriched
for 3 specific variable region (VR) sequences. Specifically, the
25-base long sequences VR1, VR2, and VR6 accounted for
20%, 13%, and 10% of the observed sequences, respectively
(Table 1). Aptamers VR11 and VR20 represented unique
sequences identified in this search that also selectively bound to
TNFα. Aptamer VR11 was the only aptamer that inhibited
human TNFα functions on cells. Interestingly, VR11
incorporates the sequence 5′-CAGTCGGCGA-3′, which was
identical to a region of aptamer VR12 with the exception of a
single G base insertion [5′-CAGGTCGGCGA-3′]. This 10base-long conserved span represents a 40% coverage of the
variable region element of our library, and despite the
homology between these two sequences, VR12 displayed no
inhibitory activity. This finding suggests that even a single base
insertion can eliminate the functional properties of such
aptamers.
In order to identify and confirm that observed sequences
were specifically binding to TNFα, 96-well ELISA microtiter
plates were coated with human TNFα and subsequently
exposed to synthetic full-length (FL; a sequence that includes a
specific VR sequence as well as the two flanking primer
regions) and variable region (VR) only DNA aptamers
harboring a 5′ biotin group. Aptamer binding to TNFα was
confirmed with streptavidin-HRP for 20 unique sequences
tested (Figure 1A) with most of them showing an ELISA signal
at least double that of the control aptamers (cApt(VR),
cApt(FL)).
DNA Aptamer VR11 Inhibits the Binding of TNFα to
Its Receptor. NF-κB (nuclear factor kappa-light-chain
enhancer of activated B cells) is a downstream eukaryotic
Figure 1. Binding of selected full-length (FL) and variable region
(VR) DNA aptamers to TNFα and NfκB assays identify FL11 and
VR11 as TNFα inhibitors. (A) Histogram of ELISA signals confirming
the binding of biotinylated FL (gray bars) and VR aptamers (black
bars) to immobilized recombinant TNFα. The control DNA aptamers
cApt(VR) and cApt (FL) gave ELISA signals comparable to those of
wells treated with streptavidin-HRP alone (open bar). (B) Histogram
of fold increase in luciferase activity in PANC-1 cells transiently
transfected with a DNA plasmid construct expressing the luciferase
gene under the control of NfκB DNA binding promoter. Cells were
treated with TNFα (100 ng/mL), FL (dark gray bars) and VR
aptamers (black bars) or an inhibitory anti-TNFα mAb (light gray
bar). Only aptamer VR11 and its full-length variant FL11 displayed
inhibitory activities comparable to that of a control anti-TNFα mAb.
(C) Histogram of TNFα-induced NF-κB luciferase activity in
HEK293T cells. Cells were treated with 2 μM (black bars), 200 nM
(gray bars), and 20 nM (white bars) of the control aptamer cApt
(VR), the TNFα specific DNA aptamers VR11, VR20, as well as 20
μg/mL of the inhibitory anti-TNFα mAb. Each histogram bar
represents the average enhancement in observed luciferase activity +
SEM (n = 9) normalized to that of untreated samples.
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Table 1. TNFα Specific DNA Aptamer Sequences Identified from the SELEX Screen As Well As Control Aptamer [cApt(VR)
and cApt(FL)] Sequences Used in the Present Study
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aptamer
sequence
VR1
VR2
VR4
VR5
VR6
VR8
VR11
VR12
VR16
VR20
cApt(VR)
FL1
FL2
FL4
FL5
FL6
FL8
FL11
FL12
FL16
FL20
cApt(FL)
ACAACCGACAAATTATCGCACTTAC
ATCACAGCGGGTACGAATGGCAGTG
ACGCTACGGGACTCTCAAACCGCC
AAGAACTGGCAGGCCGACCACCGGT
CGTCCGCTTTGAGTCTCGAAAAGGG
ATACCCATGGTACGACGGGCCATTC
TGGTGGATGGCGCAGTCGGCGACAA
CTCGTCAGTTCAGGTCGGCGATCAT
AGTGAGCGCTTAGTCTGCGCACTGG
TCCTCATATAGAGTGCGGGGCGTGT
AGTCAGTCAGTCAGTCAGTCAGTCA
AATTAACCCTCACTAAAGGGACAACCGACAAATTATCGCACTTACCTATAGTGTCACCTAAATCGTA
AATTAACCCTCACTAAAGGGATCACAGCGGGTACGAATGGCAGTGCTATAGTGTCACCTAAATCGTA
AATTAACCCTCACTAAAGGGACGCTACGGGACTCTCAAACCGCCCTATAGTGTCACCTAAATCGTA
AATTAACCCTCACTAAAGGGAAGAACTGGCAGGCCGACCACCGGTCTATAGTGTCACCTAAATCGTA
AATTAACCCTCACTAAAGGGCGTCCGCTTTGAGTCTCGAAAAGGGCTATAGTGTCACCTAAATCGTA
AATTAACCCTCACTAAAGGGATACCCATGGTACGACGGGCCATTCCTATAGTGTCACCTAAATCGTA
AATTAACCCTCACTAAAGGGTGGTGGATGGCGCAGTCGGCGACAACTATAGTGTCACCTAAATCGTA
AATTAACCCTCACTAAAGGGCTCGTCAGTTCAGGTCGGCGATCATCTATAGTGTCACCTAAATCGTA
AATTAACCCTCACTAAAGGGAGTGAGCGCTTAGTCTGCGCACTGGCTATAGTGTCACCTAAATCGTA
AATTAACCCTCACTAAAGGGTCCTCATATAGAGTGCGGGGCGTGTCTATAGTGTCACCTAAATCGTA
AATTAACCCTCACTAAAGGGAGTCAGTCAGTCAGTCAGTCAGTCACTATAGTGTCACCTAAATCGTA
transcription factor that is activated by various intra- and
extracellular cytokines, including TNFα.21 We selected a cellbased assay using an NF-κB luciferase reporter assay in order to
define aptamer sequences that inhibited human TNFα from
binding to its receptor, leading to the loss of a cell signaling
event. Our preliminary screen was conducted using the human
pancreatic cancer cell line PANC-1 transiently transfected with
the NF-κB luciferase reporter.22 The luciferase activity was
measured 6 h post-transfection with only two DNA aptamers,
namely, VR11 and FL11, displaying an ability to inhibit TNFαdependent NF-κB expression (Figure 1B). We subsequently
used a more sensitive TNFα-mediated NF-κB assay employing
HEK293T cells [higher transfection efficiency] and confirmed
the inhibitory ability of VR11 in a dose-dependent manner
(Figure 1C). The inhibition of TNFα-mediated NF-κB signal
was observed down to a VR11 aptamer concentration of 200
nM, which represented a 35-fold molar excess of aptamer over
TNFα. As expected, control aptamers VR20 and cApt(VR) did
not show any inhibition of TNFα-mediated signaling,
suggesting that the inhibitory effects of VR11 were not due
to a concentration-dependent, nonspecific binding event.
The L929 mouse fibroblast cell line was subsequently
employed to define the ability of aptamer VR11 to inhibit
human TNFα-induced cytotoxicity.23 The TNFα CD50 value
for L929 mouse fibroblast cells is approximately 3 nM (Figure
2). When L929 mouse fibroblast cells were treated with human
TNFα in the presence of aptamer VR11 or an inhibitory TNFα
mAb, the observed CD50 values were shifted to approximately
100 nM and 260 nM, respectively, representing a 33-fold and
86-fold decrease in toxicity (Figure 2). These CD50 values for
aptamer VR11 and TNFα mAb correspond to approximately a
24-fold and 5-fold molar excess over TNFα, respectively. The
control aptamer cApt(VR) as well as the TNFα-specific DNA
aptamer VR20 showed no effect on the toxicity of TNFα
(Figure 2). Besides the removal of the 3′ terminal A base of
VR11, the truncation of bases from either the 3′ or 5′ ends of
Figure 2. Aptamer VR11 inhibits TNFα-induced cytotoxicity in
murine fibroblasts. L929 cells were treated with increasing
concentrations of TNFα alone (▲) or in the presence of either the
inhibitory anti-TNFα mAb (△), inhibitory aptamer VR11 (○), the
control aptamer cApt (VR) (□), or aptamer VR20 (■). Each point
represents the average % survival value ± SEM (n = 12).
VR11 resulted in the loss of its inhibitory activity toward
human TNFα (Supplementary Figure 1).
The binding properties of two TNFα-binding DNA
aptamers, namely, VR11 and VR20, were further characterized
by surface plasmon resonance (SPR) with their binding
constants determined to be in the low nanomolar range
(respective Kd values of 7.0 ± 2.1 nM and 8.7 ± 2.9 nM). To
assess the specificity of VR11 and VR20 for human TNFα, we
also used human TNFβ as an analyte. TNFβ was chosen since
it binds to TNF receptors and is the closest known homologue
to TNFα having highly similar structures (Figure 3B) despite
having low sequence homology (Figure 3A). It was
subsequently observed that aptamers VR11 and VR20 showed
no binding to TNFβ by SPR (Figure 3E and F). One feature of
aptamer VR11 that may contribute to its inhibitory function
may be its slow on- and off-rates (Figure 3). Once bound to
TNFα, VR11 was able to block TNFα-enhanced NO
production in macrophages over a period of 24 h (Figure 4B;
as referred to as NO2− levels). Specifically, one of the
consequences of unregulated overproduction of TNFα is the
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Figure 3. Binding kinetics of TNFα to aptamers VR11 and VR20 as determined by surface plasmon resonance. (A) Sequence alignment of soluble
TNFα and the closest known homologue TNFβ. (B) Alignment of ribbon diagrams derived from the crystal structures of monomeric TNFα
(orange, PBD: 1TNF) and TNFβ (blue, PDB: 1TNR). Fitted binding curves of immobilized inhibitory aptamer VR11 associated with increasing
concentrations of (C) TNFα and (E) TNFβ (n = 3). Binding curves of immobilized aptamer VR20 associated with increasing concentrations of (D)
TNFα and (F) TNFβ (n = 3). (G) Calculated dissociation constants (KD) for TNFα binding to aptamer VR11 and VR20.
Figure 4. Inhibition of TNFα-induced NO2− production in macrophages. RAW264.7 cells were stimulated for (A) 6 h or (B) 24 h with IFNγ, IFNγ
and TNFα or IFNγ, TNFα in the presence of either the inhibitory anti-TNFα mAb, inhibitory aptamer VR11, control aptamer cApt(VR), and
aptamer VR20. The concentration of NO2− produced in culture medium was determined using the Griess assay. Each point represents the average
amount of NO2− produced ± SEM (n = 9).
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Figure 5. Structural analysis of aptamers VR11 and VR20 by circular dichroism. CD spectra of the 25-bp aptamers VR11, VR20, and cApt in the
presence of either (A) sodium or (B) potassium ions.
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persistence of inflammation. Nitric oxide (NO) is a key
mediator of inflammation stimulated by TNFα that is released
by macrophages (Figure 4). We thus used the measurement of
nitrite ions [NO2−] in solution as a marker of TNFα-induced
inflammatory signaling in the macrophage cell line RAW264.7.
Human TNFα alone does not elicit the release of nitrite ions
from macrophages. However, a synergistic effect occurs when
macrophages are concomitantly treated with interferon-gamma
(IFN-γ) and TNFα.24 At the 12 and 24 h time periods, aptamer
VR11 was able to inhibit ∼54% and ∼45% of the TNFαdependent NO release signal, respectively (Figure 4). In
comparison, the TNFα mAb blocked ∼79% and ∼74% of the
NO release signal over the same two time periods (Figure 4).
No inhibition of TNFα-induced NO release by RAW264.7 cells
was observed when treated with control aptamers cApt(VR)
and VR20. This sequence thus represents a template that may
be useful for modifications that may yield a suitable product for
use in vivo.
Structural Features of DNA Aptamer VR11. The site
targeted by VR11 on TNFα was not defined in the present
study. The epitope recognized by the mAb to TNFα has not
been identified to date, and as such this probe could not be
used to locate the VR11 binding site on TNFα. In addition, the
size and high avidity of this anti-TNFα mAb was able to block
the binding of VR4, VR11, and VR20 TNFα-directed aptamers
to their target (see Supplementary Figure 2). A structural
feature of aptamer VR11 that may contribute to its inhibitory
activity is its predicted G-quadruplex structure as determined
by the QGRS Mapper software.25 G-quadruplexes arise from
the association of four G-bases into a cyclic Hoogsteen Hbonding arrangement. Identifying G-quadruplex structures
within aptamers has been the focus of recent interest in view
of the therapeutic efficacy of previously identified G-quadruplex
forming ssDNA and RNA.26−28 A case in point is the 15-mer
DNA thrombin aptamer, which folds into a G-quadruplex
structure.29 Structurally, VR11 incorporates 11 guanine bases,
which accounts for 44% of its sequence. G-quadruplexes
typically occur when 3 or more consecutive G bases are present
within a sequence.30 However, the G-rich content of VR11
sequence displays an 18-base-long stretch containing 4 GG
repeats (5′-GGTGGATGGCGCAGTCGG-3′) that may stabilize the aptamer.31 Interestingly, the DNA aptamer KS-B,
specific for thrombin, also incorporates 4 GG repeats within its
sequence (GGTTGGTGTGGTTGG) and adopts an intramolecular G-quadruplex structure (monomeric chair form) as
proven from its crystal structure.32−34 Thus, the projected Gquadruplex for VR11 may exist in solution. An intramolecular
G-quartet structure35,36 within VR11 would support our SPR
data that suggest a 1:1 association of VR11 with TNFα. The
circular dichroism spectra of VR11, VR20, and the control
aptamer cApt were thus recorded in order to monitor for the
possible occurrence of a G-quadruplex structure within these
DNA aptamers. The sequence of VR20 harbors 4 consecutive
G bases [Table 1; 5′-CGGGGC-3′motif], and its CD spectrum
displays a negative ellipticity band centered around 240 nm as
well as a positive band near 260 nm. This spectrum indicates
that the GGGG motif present within VR20 adopts a
characteristic G-quadruplex structure in solution with a stacking
pattern involving guanosines having identical glycosidic bond
angles giving rise to a four-stranded parallel quadruplex
structure typically observed for the motif 5′-TGGGGT3′.37,38 The CD spectrum of control aptamer cApt did not
reveal any known spectral features indicative of G-quadruplexes. As for VR11, its CD spectrum harbors a broad
positive band around 290 nm, suggesting that guanosines with
different glycosidic bond angles within its sequence are stacked
in the presence of sodium or potassium (Figure 5A and B).
Unlike the CD spectrum observed for VR20, the VR11
spectrum does not provide conclusive proof that the guanosine
elements within its 25-bp sequence are arranged in a traditional
G-quadruplex structure. Nevertheless, a structural arrangement
involving the stalking of guanosine bases is present in VR11
and may stabilize its structure. Aptamer stability is crucial for
targeting TNFα since the effectiveness of current protein-based
therapies can be partly attributed to their long circulating halflives, such as Etanercept, which is linked to the Fc portion of an
IgG1 to help extend its half-life to 4−5 days, or the PEGylation
of anti-TNF antibodies.39,40 Recently, some of the most
common modifications of aptamers investigated have been
their conjugation to poly(ethylene glycol) (PEG) groups for
increased stability.41,42 In addition, to increase the in vivo
circulating half-life of DNA aptamers, modified nucleotides
could be introduced into the libraries during synthesis such as
2′-OH group with 2′-fluoro or 2′-amino, aromatic, or alkyl
moieties or introduction of locked nucleic acid (LNA).43,44
Also, aptamer libraries with longer variable regions may identify
sequences with a higher G-rich content, which in turn could
adopt different G-quadruplex structures and yield improved
inhibitors to TNFα.45
Immunogenicity of DNA Aptamer VR11. One therapeutic obstacle that DNA aptamers may encounter is their
possible activation of an immune response similar to that of
oligodeoxynucleotides containing CpG motifs. These CpG
motifs can promote a Th1 response by signaling through TLR9,
which is expressed on human B cells and plasmacytoid
dendritic cells leading to an innate immune response.46,47
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Figure 6. Aptamer VR11 does not cause an innate immune response in vivo. (A) Potential CpG motifs located in the variable region of aptamer
VR11. C57BL/6 mice (n = 3) were given an intraperitoneal injection of 10 μg of CpG ODN, 100 μg of aptamer VR11 or cApt, and the amount of
(B) TNFα or (C) KC in serum 3 h after injection was determined by ELISA.
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performed using the L929 cell line sensitized with actinomycin D and
compared to a commercial recombinant human TNFα purchased from
Bioclone Inc. (Supplementary Figure 3).
Aptamer Selection Screens. The initial ssDNA library contained
a central randomized sequence of 25 nucleotides flanked by T3 and
SP6 primer regions, respectively. Briefly, the sequence of the starting
library was 5′ AAT TAA CCC TCA CTA AAG GG-(25N)-CTA TAG
TGT CAC CTA AAT CGTA. The forward primer 5′ AAT TAA CCC
TCA CTA AAG GG 3′ and reverse primer 5′ TAC GAT TTA GGT
GAC ACT ATA G 3′ were used for selection and cloning (IDT
Technologies, Inc.). To begin the selection, a 50 nmol aliquot of the
library representing ∼3.0 × 1016 molecules consisting of ∼25 copies of
each possible unique sequence was first counter-selected against a 6His peptide (HHHHHH) loaded onto Ni-NTA magnetic agarose
beads. The resulting sub-library was then exposed to 10 μg of Histagged TNFα immobilized onto Ni-NTA beads suspended in
Selection Buffer (PBS, 0.005% (v/v) Tween-20) at 37 °C for 1 h.
Unbound DNA sequences were washed away (PBS, 0.005% (v/v)
Tween-20), and DNA−protein complexes were eluted from the
recovered beads using an imidazole buffer (PBS, 0.005% (v/v) Tween20, 240 mM imidazole). The ssDNA component was precipitated with
sodium perchlorate/isopropanol and recaptured using a silica
membrane-based purification system (Qiagen Inc.). The DNA
aptamers were then amplified by asymmetrical PCR using a 100-fold
excess of forward primer. After every three subsequent rounds of
selection, the amount of target was reduced in half to increase the
selection pressure to capture the tightest binding species. After 12
rounds of selection, the bound sequences were amplified by PCR,
cloned into a pCR4-TOPO TA vector (Invitrogen), and analyzed
using BioEdit sequence alignment editor software (Ibis Therapeutics).
Aptamer-Based Enzyme Linked Binding Assay. A 96-well
ELISA microtiter plate (BD Falcon) was coated overnight at 4 °C with
100 μL of human TNFα (10 μg/mL) prepared in coating buffer (0.2
M carbonate/bicarbonate, pH 9.4). The plate was then washed 3 times
with PBS-T (0.1 M phosphate, 0.15 M sodium chloride, pH 7.0
containing 0.05% Tween-20) and blocked overnight at 4 °C in 150 μL
of blocking solution (PBS-T + 1% (w/v) BSA). The plate was washed
3 times with PBS-T and incubated overnight at 4 °C with 100 μL (10
μg/mL) of 5′ biotinylated aptamers dissolved in PBS, 0.005% (v/v)
Tween-20. The plates were washed and incubated for 1 h at 4 °C with
100 μL of streptavidin-HRP (1:2000). Plates were then read on a plate
reader at 450 nm following washing and dispensing of 100 μL of TMB
substrate (1 TMB tablet (Sigma-Aldrich Inc.) dissolved in 1 mL of
DMSO in 10 mL of 0.05 M phosphate citrate, pH 5.0 and 2 μL
sodium peroxide) per well. Color development was stopped by adding
50 μL of 1 M sulphuric acid per well.
NF-κB Luciferase Reporter Assay. An NF-κB luciferase reporter
plasmid was transfected into cells as a marker of TNFα binding as
previously described.22 Briefly, 0.1 μg/well TA-LUC NF-κB and 10
VR11 contains two possible CpG motif sequences (Figure 6A).
To test whether this DNA aptamer can induce an innate
immune response, we injected 100 μg of aptamer VR11 and
cApt as well as 10 μg of a known TLR9 ligand CpG ODN in
the intraperitoneal cavity of C57BL/6 mice and quantified the
amount of TNFα and mouse IL-8 present in their serum after 3
h. The CpG ODN positive control yielded a 35-fold increase in
TNFα and a 24-fold increase in murine IL-8 serum levels, while
the control aptamer cApt (which contains no CpG motifs) and
the TNFα-specific VR11 DNA aptamer yielded statistically
nonsignificant increases in both TNFα and murine IL-8
cytokine levels as compared to untreated mice (Figure 6B
and C).
Summary. Short DNA oligonucleotides such as aptamers
are considered to be nonimmunogenic. This study also
establishes that VR11 does not trigger an innate immune
response associated with the production of inflammatory
cytokines (Figure 6), which is relevant since TNFα antagonists
are now well established as an effective treatment for an array of
inflammatory disorders in which TNFα plays an important
pathological role. Current protein-based therapies have been
effective, although these agents have now been shown to
display immunogenicity following their long-term use. As well,
a significant proportion of patients with inflammatory disorders
are nonresponders to such protein-based therapeutics.48
Although DNA-based aptamers are small, synthetic targeting
agents, they still face several obstacles to overcome as
therapeutic agents. Aptamer VR11 does provide a simple
scaffold for developing DNA-based inhibitors of TNFα with
pharmacological properties distinct from those of existing
classes of therapeutic agents aimed at treating inflammatory
diseases.
■
METHODS
Expression and Purification of Human TNFα. The expression
plasmid (RCA-093) encoding a soluble 157-amino acid long, Histagged human TNFα recombinant protein (residues 77−233) was
obtained from Bioclone Inc. The protein was expressed in Escherichia
coli BL21 (DE3) strain. The expression of the TNFα (77−233)
fragment was induced in culture overnight with isopropyl β-Dthiogalactopyranoside (IPTG, 0.5 mM). Soluble human TNFα was
purified by nickel affinity chromatography under nondenaturing
conditions. Purified human TNFα was characterized by SDS-PAGE
and quantified using the Bradford assay.49 To verify that the protein
was properly folded and functionally active, a cytotoxicity assay was
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ng/well β-gal CMV were co-transfected to the cells. PANC1 (ATCC)
(1.0 × 104) and HEK293T (ATCC) (5.0 × 103) cells were seeded in a
96-well plate 24 h before the transfection steps. DNA was transfected
into cells using Lipofectamine 2000 (Invitrogen Canada Inc.). Cells
were incubated 4−6 h in Opti-mem medium (Promega) and
transferred to 10% FBS DMEM overnight. Aptamers were added to
the medium (1 μM final concentration for PANC-1 cells or 2 μM, 200
nM, and 20nM for HEK293T) 30 min prior to treating the transfected
cells with recombinantly expressed human TNFα (100 ng/mL).
Luciferase activity was measured after 6 h using the Dual-Light System
luciferase assay from Applied Biosystems according to the
manufacturer’s protocol and read on a Luminoskan Ascent
luminometer (ThermoLab Systems). The results were normalized to
the values obtained for β-galactosidase activity.
Inhibition of TNFα Induced Cytotoxicity. L929 cells (ATCC)
were seeded 24 h before experiments in 96-well flat-bottom microtiter
plates at a density of 1.0 × 104 cells/well in DMEM medium
containing 10% FBS. Aptamers (2 μM) or 20 μg/mL anti-TNFα mAb
(R&D systems) were incubated with human TNFα for 2 h in PBS
prior to incubation with cells. Subsequently, aptamer-TNFα samples
were added to the cells for 2 h. Cells were then washed with warm PBS
and incubated in complete medium for another 48 h. The viability of
adherent cells was subsequently determined using the sulforhodamine
B assay.50
Surface Plasmon Resonance. All experiments were performed
using the ProteOn XPR36 protein interaction array system (Bio-Rad
Laboratories, Inc.) and one ProteOn NLC sensor chip, coated with
NeutrAvidin for coupling of biotinylated molecules. Biotinylated
aptamers VR11, VR20, and rApt(VR) were synthesized with a 5′
biotin with a standard C6 spacer and HPLC purified (Integrated DNA
Technologies, Inc.). Subsequently 2.5 nM of aptamer was diluted in
PBS-T and injected for 30 s at 30 μL/min. Aptamer loading on the
chip yielded approximately 10−12 response units, which represented
∼1 ng of aptamer. Human TNFα and TNFβ stock solutions were
diluted as a series of 2-fold dilutions ranging from 2 μM to 3.9 nM in
PBS-T (0.05% (v/v) Tween-20) pH 7.4 (Figure 3C−F). Protein
concentrations and a buffer control were injected in the analyte
channel with a contact time of 120 s, dissociation time of 800 s, and a
flow rate of 100 μL/min. The ligand channels were regenerated with a
30 s injection of 1 M H3PO4 followed by a 30 s injection of 1 M NaCl.
All experiments were performed at 25 °C and repeated in triplicate.
Sensorgrams were then double-referenced by subtracting the buffer
response and using the interspot reference. The sensorgrams were
fitted globally to a 1:1 Langmuir binding model, and the kinetic
parameters for the association (ka), dissociation rates (kd), and binding
constant (KD) were derived from the fitted curves.
Determination of Aptamer Concentration and Circular
Dichroism Spectroscopy. The oligonucleotide concentration of
each aptamer solution was determined from its absorbance at 260 nm
as measured at 25 °C with a Cary 300 Bio spectrophotometer (Varian
Canada, Inc.) using molar extinction coefficients of 244 400 M−1 cm−1,
239 400 M−1 cm−1, and 251 800 M−1 cm−1 for VR11, VR20, and cApt
respectively. Circular dichroism (CD) spectra were recorded at 25 °C
using an Aviv model 62 DS spectropolarimeter (Aviv Associates). The
buffer used for CD measurements contained 10 mM phosphate, 0.1
mM EDTA (free acid), and 100 mM of either sodium or potassium
ions. Either KOH or NaOH was added to the buffers until pH 7 was
reached, and the ionic strengths of the buffers were adjusted to the
desired level by adding known amounts of NaCl or KCl. Dry and
desalted DNA aptamers were dissolved in the buffers until a
concentration of ∼0.03 mM was achieved. The DNA aptamer
solutions were heated to 100 °C for 5 min to transform the DNA
aptamer into their unfolded forms and slowly allowed to cool to 25 °C.
Ellipticity values (reported as θ) were measured from 320 to 200 nm
in 1 nm increments using a quartz cuvette with an optical path length
of 0.1 cm.
Inhibition of TNFα-Induced NO Production in Macrophages.
Experiments were modified using a previous protocol.51 Briefly, RAW
264.7 cells (ATCC) were seeded at a density of 1.0 × 105 cells/well in
a 12-well plate in RPMI 1640 + 10% FBS. Cells were pretreated for 1 h
with 2 U/mL IFN-γ (Peprotech) and then treated with 100 ng/mL of
human TNFα in 1 mL of medium with aptamers VR11, VR20, or the
control aptamer cApt(VR) at a final concentration of 2 μM or with 10
μg/mL anti-TNF mAb. Aliquots of the medium [100 μL] were
removed at each time point, and the NO2− level was determined using
the Griess reagent kit for nitrite determination (Invitrogen).
Analysis of VR11 Ability To Activate an Innate Immune
Response. The aptamer VR11 and cApt were dissolved in sterilized
saline at a concentration of 500 μg/mL, and CpG ODN (5′TCCATGACGTTCCTGACGTT-3′; type B murine, ODN 1826,
Invivogen) was dissolved at a concentration of 50 μg/mL. Intraperitoneal injections (200 μL) were administered in C57BL/6 mice.
The untreated group received an injection of 200 μL of saline alone.
Three hours after injection, mice were sacrificed, and their serum was
collected for analysis. Protein cytokine concentrations were
determined using the DuoSet ELISA development kits for mouse
CXCL1/KC (murine IL-8, R & D systems Inc.) and murine TNFα (R
& D systems Inc.) as per manufacturers protocols. Animals were kept
under standard pathogen-free conditions at the Ontario Cancer
Institute animal facility. Experiments were performed in accordance
with the rules and regulations of the Canadian Council for Animal
Care.
■
ASSOCIATED CONTENT
S Supporting Information
*
This material is available free of charge via the Internet at
http://pubs.acs.org.
■
AUTHOR INFORMATION
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476
Corresponding Author
477
*E-mail: [email protected].
478
Notes
479
The authors declare no competing financial interest.
480
■
ACKNOWLEDGMENTS
We wish to thank S. Mamaghani and D. Hedley for providing
access to TA-LUC NF-κB, β-gal CMV, and PANC1 cells. We
would also like to thank T. Watts for providing the HEK293T
cells and also K. Geddes and D. Philpott for help with immune
monitoring assays. This work was supported by an operating
grant from the Canadian Breast Cancer Foundation (J.G.) and
a studentship award to E.W.O. from the Canadian Institutes of
Health Research Training Program in Biological Therapeutics.
■
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